JP5770775B2 - Stable pharmaceutical composition containing factor VIII - Google Patents

Description

  The present invention relates to a stable pharmaceutical composition containing factor VIII.

  Factor VIII is a well-known plasma protein that is essential for the blood clotting process and is therefore used in the treatment of hemophilia.

  Various forms of Factor VIII have been used or are intended for use as active ingredients for the treatment of hemophilia. These factor VIII include human factor VIII [Humate® P, Monoclate® P, Immunate® or Hemofil® M active ingredient, etc.], recombinant human factor VIII Factor VIII [r-VIIISQ (ReFacto® active ingredient) or Kogenate® or Recombinate® active ingredient etc. described in PCT patent application WO 91/09122], porcine factor VIII [A product sold by Ipsen Inc., USA, Hyate: C® active ingredient] or recombinant porcine factor VIII [eg described in patent application WO 01/68109 and identified as “POL1212” Denatured B-domainless porcine factor VIII or the protein of SEQ ID No 38 of the above-mentioned patent application].

  The stability of the formulation has always been a problem for the pharmaceutical industry dealing with Factor VIII pharmaceutical compositions.

  Albumin is often used to stabilize these formulations. However, despite its interesting stabilizing effect, albumin has the disadvantage of being expensive and risking carrying infectious organisms such as prions. For these reasons, in the pharmaceutical industry, albumin has recently been replaced with other stabilizers in Factor VIII pharmaceutical compositions.

  Various albumin-free pharmaceutical compositions are already known to those skilled in the art. For example, U.S. Pat.No. 5,565,427 describes Factor VIII: C, amino acids or salts or homologues thereof and detergents (e.g. polysorbate 80 or Tween® 80) or organic polymers (e.g. polyethylene). A stabilized albumin-free solution having Factor VIII: C activity is taught.

  U.S. Pat.No. 5,605,884 describes a stable consisting of Factor VIII and a high ionic strength medium--which preferably consists of an aqueous solution containing a mixture of sodium chloride, calcium chloride and histidine as buffer ions. Relates to a factor VIII composition.

  U.S. Pat. ing.

  US Pat. No. 5,962,650 includes recombinant factor VIII, calcium salts such as calcium chloride and preferably antioxidants, nonionic surfactants, sodium chloride or potassium, amino acids and mono- or disaccharides. A stable albumin-free recombinant factor VIII composition comprising an aqueous solution having a low oxygen concentration is disclosed.

  U.S. Pat.No. 5,972,885 describes high concentrations (at least 1,000 IU / ml) of recombinant factor VIII, and preferably (especially) sodium chloride or potassium, calcium chloride, nonionic surfactants [e.g. Poloxamer, mono- or disaccharide (preferably sucrose) and an antioxidant (eg citric acid), one or more components selected from the group consisting of subcutaneous, intramuscular or intradermal It relates to a pharmaceutical preparation for administration.

  PCT patent application WO 89/09784 includes a gel of a buffer solution containing said factor VIII and tris (hydroxymethyl) methylamine, trisodium citrate, sodium chloride, sucrose and calcium chloride A process for the production of a thermostable factor VIII concentrate is disclosed which comprises filtration and subsequent lyophilization of the resulting concentrate. The factor VIII thus produced can withstand temperatures up to 80 ° C. for up to 72 hours.

  PCT patent application WO 94/07510 discloses a factor VIII composition stabilized by a nonionic surfactant [eg, a poloxamer such as polysorbate 80]. Such compositions may also contain (especially) one or more components selected from the group consisting of mono- or disaccharides such as sodium chloride or potassium, calcium chloride, amino acids, sucrose.

International Publication No. WO91 / 09122 International Publication No. WO01 / 68109 U.S. Pat.No. 5,565,427 U.S. Pat.No. 5,605,884 US Patent No. 5,763,401 U.S. Pat.No. 5,874,408 US Patent No. 5,962,650 U.S. Pat.No. 5,972,885 International Publication WO89 / 09784 International Publication No. WO94 / 07510

The inventor has now unexpectedly found that the following ingredients:
(a) Factor VIII;
(b) a surfactant;
(c) calcium chloride;
(d) sucrose;
(e) sodium chloride;
(f) trisodium citrate; and
(g) a buffer containing no amino acids;
A solid pharmaceutical composition obtained by lyophilization of a solution containing no amino acids and having a pH of 6 to 8 before lyophilization and after reconstitution in water for injection It was discovered that the objects also show stability over time.

  As used herein, factor VIII is human factor VIII, recombinant human factor VIII, porcine factor VIII, recombinant porcine factor VIII, or more generally, Means another recombinant factor VIII that can be used instead of

  It will be preferred that the Factor VIII contained in the composition of the invention is selected from porcine factor VIII and recombinant porcine factor VIII. More preferably, the factor VIII contained in the composition according to the invention is recombinant porcine factor VIII, in particular a modified B-domainless form as disclosed in patent application WO 01/68109. domainless form) porcine factor VIII, ie, a modified porcine factor VIII having the amino acid sequence of SEQ ID No 1 shown below:

  It will be preferred that the surfactant is a non-ionic surfactant. Nonionic surfactants include block copolymers such as polysorbates and poloxamers (ie, copolymers of polyethylene and propylene glycol), among others. According to a preferred modification of the invention, the surfactant is a polysorbate. More preferably, the polysorbate contained in the composition of the present invention has an average degree of polymerization of 20 to 100 monomer units (preferably about 80), and may be, for example, polysorbate 80. Preferably, the polysorbate should also be of plant origin.

  Preferably, the amino acid free buffer is tris (hydroxomethyl) methylamine (hereinafter abbreviated as “Tris”).

  Further, the pH of the pharmaceutical composition before lyophilization and after reconstitution in water for injection is preferably 6.5 to 7.5, more preferably about 7.0.

Preferably, the solid pharmaceutical composition of the present invention is
(a) Concentrations of 50-10,000 international units / ml for human factor VIII or recombinant human factor VIII or 50-10,000 pig units (pig factor VIII for porcine factor VIII or recombinant porcine factor VIII Factor VIII at a concentration of (porcineunit) / ml;
(b) a surfactant having a concentration of not less than the critical micelle concentration to 1 v / v%;
(c) calcium chloride at a concentration of 0.5-10 mM;
(d) Sucrose at a concentration of 5-50 mM
(e) sodium chloride at a concentration of 0.15-0.5M;
(f) trisodium citrate at a concentration of 1-50 mM; and
(g) an amino acid free buffer at a concentration of 1-50 mM;
It was obtained by lyophilization of a solution containing no amino acids.

  In order to assess activity with respect to international factor VIII unit, the product to be tested should be a Concentrate standard such as the British Standard NIBSC 95/608 (NIBSC for National Institute of Biological Standards and Control). Standard).

  Porcine unit of factor VIII means the United Kingdom national standard maintained by the NIBSC in the UK. In order to assess the activity with respect to porcine factor VIII unit, the product to be tested is assessed against the UK national porcine standard NIBSC 86/514. With respect to recombinant porcine factor VIII, it should be understood that one unit of activity for recombinant porcine factor VIII is equivalent to one unit of activity for porcine factor VIII.

The solid composition of the present invention comprises the following components:
• A factor of 100-5,000 international units / ml for human factor VIII or recombinant human factor VIII or a concentration of 100-5,000 pig units / ml for porcine factor VIII or recombinant porcine factor VIII Factor VIII;
A surfactant at a concentration of 0.002 to 0.04 v / v%;
-Calcium chloride at a concentration of 1-5 mM;
Sucrose at a concentration of 5 to 25 mM sodium chloride at a concentration of 0.2 to 0.4 M;
• Trisodium citrate at a concentration of 1-20 mM; and • A buffer without amino acids at a concentration of 1-20 mM;
More preferably, it is obtained by lyophilization of a solution containing at least one of the above and not containing amino acids.

The solid composition of the present invention has the following properties:
A concentration of 200-2,000 international units / ml (particularly about 1,000 international units / ml) for human factor VIII or recombinant human factor VIII or 200 for porcine factor VIII or recombinant porcine factor VIII Factor VIII at a concentration of ˜2,0000 pig units / ml (especially about 1,000 pig units / ml);
A surfactant at a concentration of 0.002 to 0.02 v / v% (especially about 0.01 v / v%);
-Calcium chloride at a concentration of 1-3 mM (especially about 2 mM);
-Sucrose at a concentration of 5-15 mM (especially about 11.7 mM)-Sodium chloride at a concentration of 0.25-0.35 M (especially about 0.3 M);
• Trisodium citrate at a concentration of 1-20 mM (especially about 10 mM); and • A buffer without amino acids at a concentration of 5-15 mM (especially about 10 mM);
More preferably, it is obtained by lyophilization of an amino acid-free solution having at least one of the following.

  The solid factor VIII composition of the present invention comprises a suitable amount of the components identified as (a), (b), (c), (d), (e), (f) and (g). It can be prepared by lyophilization using standard techniques (such as sterilization).

  The stability of a solid composition over a period of time may be measured, for example, by the methods described herein under the heading “Analytical Methods” or other methods recognized by those skilled in the art. obtain.

  The compositions of the present invention are evaluated using the method described in the section entitled “Analytical Methods” herein, 70% to 130% (preferably 80 to 120%) of the original factor VIII activity. ) Is held stable over a period of time, it is considered stable over a period of time.

  The solid composition of the present invention is preferably stable for at least 6 months or 12 months when held at a temperature of 2-8 ° C. More preferably, it is stable for at least 6 months or 12 months when held at a temperature of 30-32 ° C.

  The solid factor VIII composition of the present invention can be diluted with sterile water, optionally containing sodium chloride, and then the resulting liquid pharmaceutical composition can be directly injected into a patient in need thereof. A liquid pharmaceutical composition obtained by diluting the obtained liquid pharmaceutical composition and the solid factor VIII composition of the present invention with sterilizing water containing sodium chloride, if necessary, also forms part of the present invention.

  A method for treating hemophilia comprising administering the liquid composition of the present invention to a patient in need thereof is also encompassed within the scope of the present invention. The intended method of administration for the liquid composition of the present invention will preferably be intravenous administration. The dosage of the liquid composition of the present invention to be administered will be determined by a physician or veterinarian taking into account the severity of the illness of each patient.

  The term “about” means an interval near the numerical value being considered. As used herein, “about X” means an interval from 10% of X−X to 10% of X + X, preferably from 5% of X−X to 5% of X + X.

  Technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs unless defined otherwise. Similarly, all publications, patent applications, all patents, and other references mentioned herein are hereby incorporated by reference.

  The following examples illustrate the above and are not intended to limit the scope of the present invention.

Example 1:
A solution was prepared containing the following ingredients in 0.5 ml of sterile water:

  The mixture was lyophilized in a sterile bottle and then sealed. The resulting solid composition was tested and shown to be stable for at least 18 months at a temperature of 2-8 ° C and at least 6 months at a temperature of 30-32 ° C when tested by Factor VIII activity. When evaluated by size exclusion HPLC (SECHPLC), no high molecular weight components were formed, and no fragment was observed when evaluated by SDS PAGE.

  The resulting lyophilized mixture will be reconstituted, for example, with 1.0 ml of sterile water prior to injection into the patient.

Example 2:
A solution containing the following ingredients in 1.0 ml of sterile water was prepared:

  The mixture was lyophilized in a sterile bottle and then sealed.

  The resulting lyophilized mixture is typically reconstituted with 2.0 ml of sterile water prior to injection into the patient.

Example 3:
A solution was prepared containing the following ingredients in 0.5 ml of sterile water:

  The mixture was lyophilized in a sterile bottle and then sealed.

  The resulting lyophilized mixture is typically reconstituted with 1.0 ml of sterile water prior to injection into the patient.

Analytical method chromogenic assay
Factor VIII activity was measured by a modified dye test (Technochrom FVIII: C Reagent Kit, Technoclone). Generation of activated factor X by factor IX was stimulated by factor VIII, which acted as a cofactor during the reaction. Release of p-nitroaniline from the chromogenic substrate was catalyzed by activated factor X. The amount of p-nitroaniline released was measured photometrically at 405 nm and a linear relationship was obtained between the amount of p-nitroaniline produced by this measurement and factor FVIII.

SECHPLC
Soluble polymer components and fragments were measured by gel filtration on HPLC using a TohoHass TSK G3000 SWXL (Waters LC Module 1 plus) equipped with a fluorescence detector and having a 0.78 × 30 cm pre-packed column. Excitation wavelength 280 nm and emission wavelength 340 nm. The result was evaluated by integration of the peak area.

SDE PAGE test
SDE PAGE (polyacrylamide gel electrophoresis using a flatbed electrophoresis device (Multiphor II LKB) and pre-cast 7.5% gel (EXCELGEL SDS, Pharmacia)) was used to measure the disruption products of the FVIII molecule. . The protein band was visualized by Coomassie blue staining.

Stability Testing Stability can be assessed by performing the above assessment at different times on the same sample held at a selected temperature (this temperature can be about + 4 ° C .; or + 31 ° C.). A composition is considered to have lost its stability when its factor VIII activity drops by 30% or more.

Claims (12)

(a) Concentration of 50-10,000 international units / ml for human factor VIII or recombinant human factor VIII or 50-10,000 pig units / ml for porcine factor VIII or recombinant porcine factor VIII Factor VIII of
(b) a surfactant having a concentration of not less than the critical micelle concentration to 1 v / v%;
(c) calcium chloride at a concentration of 0.5-10 mM;
(d). 5 to a concentration of 25 mM sucrose;
(e) sodium chloride at a concentration of 0.15-0.5M;
(f) trisodium citrate at a concentration of 1-50 mM; and
(g) an amino acid free buffer at a concentration of 1-50 mM;
A solid pharmaceutical composition obtained by lyophilization of a solution containing no amino acids and having a pH of 6-8 before lyophilization and after reconstitution in water for injection. The solid pharmaceutical composition according to claim 1, wherein the factor VIII is selected from porcine factor VIII or recombinant porcine factor VIII. A solid pharmaceutical composition according to claim 2 , wherein the factor VIII is recombinant porcine factor VIII. The solid pharmaceutical composition according to claim 2 , wherein the recombinant porcine factor VIII has the amino acid sequence of SEQ. The solid pharmaceutical composition according to one of claims 1 to 4 , wherein the surfactant is a polysorbate. The solid pharmaceutical composition according to claim 5 , wherein the surfactant is polysorbate 80. Buffer without amino acids is tris (hydroxymethyl) methylamine, the solid pharmaceutical composition according to one of claims 1 to 6. Having a pH of 6.5 to 7.5 after reconstitution in water for lyophilization before and injection, the solid pharmaceutical composition according to one of claims 1 to 7. (a) Concentration of 100-5,000 international units / ml for human factor VIII or recombinant human factor VIII or 100-5,000 pig units / ml for porcine factor VIII or recombinant porcine factor VIII Factor VIII of
(b) a surfactant at a concentration of 0.002% to 0.04%;
(c) calcium chloride at a concentration of 1-5 mM;
(d) Sucrose at a concentration of 5-25 mM
(e) Sodium chloride at a concentration of 0.15-0.4M;
(f) trisodium citrate at a concentration of 1-20 mM; and
(g) an amino acid free buffer at a concentration of 1-20 mM;
A solid pharmaceutical composition according to one of claims 1 to 8 , obtained by lyophilization of a solution containing no amino acids. The solid pharmaceutical composition according to one of claims 1 to 9, the liquid pharmaceutical composition obtainable after dilution with sterile water. The liquid pharmaceutical composition according to claim 10 , further comprising sodium chloride. A solution as defined in one of claims 1 to 9 , which does not contain amino acids.